[Choice of primers: a determining element in molecular diagnosis of cutaneous leishmaniasis]. / Choix des amorces: élément déterminant dans le diagnostic moléculaire de la leishmaniose cutanée.

The cutaneous leishmaniasis (CL) is a parasitic disease which represents a serious problem for the public health not only in Tunisia but also all over the world. Its diagnosis is based on the techniques which are usually used, direct examination and in vitro culture. Because of several factors, these techniques lack sensitivity. The molecular biology, which is indeed more rapid and more sensitive, has proved its effectiveness in diagnosis of the CL. There are two main aims for our research work. First, to show the contribution of the Polymerase Chain Reaction (PCR) during the diagnosis of CL (of course by comparing the results obtained when using this technique with those found through the direct examination); second, to compare the two pairs of primers which amplify the leishmanien gene coding for the 18s ribosomal sub-unit: the pair R221/R332 (PCR1) and the pair Lei70L/Lei70R (PCR2). Our work was carried out upon 299 samples. One hundred and eighty-eight of them were positive using the direct examination and/or the PCR and 111 were negative. Only two samples were positive using of course the direct examination in comparison with 74 which were positive when using only the PCR (PCR1 and/or PCR2). Among these 74 samples, 64 where positive using only PCR2 in comparison with two samples which were positive using only PCR1. The eight remaining samples were at once positive for the PCR1 and the PCR2. The PCR (notably the PCR2) has proved a more significant percentage of positivity in comparison with direct examination: 98.98% for the PCR and 60.6% for direct examination.

[Identification of Enterocytozoon bieneusi by PCR in stools of Tunisian immunocompromised patients]. / Identification d'Enterocytozoon bieneusi par PCR dans les selles des patients immunodéprimés tunisiens.

INTRODUCTION: Intestinal microsporidiosis is recognised as an important cause of opportunistic parasitosis in immunocompromised patients, especially HIV-infected patients. Enterocytozoon bieneusi is the common causal agent. The diagnosis of intestinal microsporidiosis has usually based on microscopic detection of the spores of microsporidia species in stool samples, requires additional staining techniques as Modified Weber's trichrome stain. However, the detection of the spores can be difficult and species determination, which is important for defining the appropriate treatment, is impossible. Polymerase chain reaction (PCR)-based methods have been successfully used for detection of microsporidian infections. They are more sensitive and are able to identify microsporidia species. The purpose of this study is to identify E. bieneusi to adapt treatment and assess the true prevalence of the intestinal microsporidiosis due to this species in compromised patients in Tunisia. PATIENTS AND METHODS: One hundred and eighteen stools from immunocompromised patients, with a symptomatology in favour of the intestinal microsporidiosis, were analysed using light microscopy after staining with Modified Weber's trichrome stain and PCR. RESULTS: Only four were positive by Modified Weber's trichrome stain whereas eleven stools were positive by PCR, giving a prevalence of 20% in HIV-infected patients and 5,35% in human immunodeficiency virus-negative patients. CONCLUSION: This study confirms the usefulness of PCR in the diagnosis of the intestinal microsporidiosis due to E. bieneusi. Indeed, PCR has greater sensitivity than Modified Weber's trichrome stain and can identify the species of microsporidia in order to adapt the treatment.

[Dermatobia hominis furuncular myiasis in a man returning from Latin America: first imported case in Tunisia]. / Myiase furonculeuse à Dermatobia hominis au retour d'Amérique du Sud: premier cas importé en Tunisie.

Human cutaneous myiasis is a common dermatosis in tropical zones. The purpose of this report is to describe the first imported case of furuncular myiasis caused by Dermatobia hominis (human botfly) in Tunisia. The patient was a man returning from Bolivia. Furuncular myiasis was suspected based on epidemiological data and clinical examination showing pruriginous elevated lesions. Diagnosis was confirmed by identification of Dermatobia hominis larvae. Treatment was based mainly on manual removal of larvae. Since furuncular myiasis is unknown in Tunisia, it is important to remember this parasitic disease in differential diagnosis in patients presenting boil-like inflammatory papules following travel to Latin America.

[Epidemiological and clinical characteristics of intestinal microsporidiosis]. / Caractéristiques épidémiologiques et cliniques de la microsporidiose intestinale chez les immunodéprimés vus à Tunis.

INTRODUCTION: Intestinal microsporidiosis is an opportunistic parasitological infection affecting mainly immunocompromised patients, particularly those infected with HIV. PURPOSE: The purpose of this study was to analyse the epidemiological and clinical characteristics of intestinal microsporidiosis and the treatments available for it. MATERIAL AND METHODS: This retrospective study examined records collected over a 13-year period (from January 1995 through December 2007). It included 572 immunocompromised patients (279 HIV-infected patients and 293 without HIV infection) with symptoms suggesting intestinal microsporidiosis. All were tested systematically for microsporidia spores by modified (Weber's) Trichrome staining. RESULTS: Fourteen patients (10 men, 4 women) were diagnosed with intestinal microsporidiosis, for a prevalence of 2.4% overall, 3.6% in HIV-infected patients and 1.4% in those without HIV infection. Intestinal microsporidiosis affected 10 HIV-infected patients, 70% of whom had a CD4 count <100 cells/mm3. Their mean age was 30+/-15 years (range: 15 months to 48 years). The average age of HIV-infected patients (36 years) was significantly higher than of those without HIV infection (15 years). Thirteen patients had symptoms, most frequently diarrhea (11 cases), sometimes associated with dehydration (5 cases). Eight patients (57%) received only symptomatic treatment, and 4 (28.6%) received albendazole. No treatment was recommended in 2 cases (14.3%). Clinical course was marked by improvement in 6 cases, death in 5, and persistence of asymptomatic carriage in one. Two patients were lost to follow-up. CONCLUSION: Intestinal microsporidiosis is a parasitological disease that mainly affects AIDS patients with CD4 counts <100 cells/mm3. Its diagnosis requires special techniques. Its symptomatology is dominated by chronic diarrhea that can cause dehydration. Effective treatment requires identification of the species.

[Pnemocystis jiroveci pneumonia: Comparison between conventional PCR and staining techniques]. / Pneumocystose à Pneumocystis jiroveci : étude comparée de la PCR et des techniques de coloration.

Diagnosis of pneumocystis pneumonia is usually based on clinical features and X-rays photography and confirmed in the laboratory by visualisation of Pneumocystis organisms in stained preparations of respiratory specimens using several techniques (Gomori-Grocott, May-Grünwald Giemsa, bleu de toluidine O). Actually, PCR has considerably increased sensitivity of detection of Pneumocystis. The aim of this study is to compare conventional PCR results to those of staining techniques (Gomori-Grocott, May-Grünwald Giemsa) in addition to the X-ray and clinical findings in order to evaluate the contribution of each method. Sixty-four respiratory specimens were collected from 54 immuno-compromised patients with clinical symptoms of pulmonary infection. We diagnosed pneumocystis pneumonia in 16 patients according to staining techniques and/or typical clinical and radiological findings and/or response to treatment. Of the 15 patients, 14 were positive by PCR and only five were positive by direct examination, yielding a sensitivity and specificity of 93.3 and 87.1% for PCR and 33.3 and 100% for staining techniques. Conventional PCR provides a sensitive and objective method for the detection Pneumocystis jiroveci from less invasive sample.

[Rhino-orbito-cerebral mucormycosis in the diabetic: a better known pathology in Tunisia]. / La mucormycose rhino-orbito-cérébrale chez le diabétique: une pathologie mieux connue en Tunisie.

This is a retrospective study including 17 patients with rhino-orbito-cerebral mucormycosis diagnosed in a period of 16 years, between 1992 and 2007, in 8 men and 9 women. All patients were diabetic with ketoacidosis diabetes in 8 cases. Necrosis facial and ophthalmic symptoms were the most frequent presenting manifestations. The diagnosis was confirmed by mycological examination, with or without histopathology, identifying Rhizopus oryzae in 12 cases. Treatment consisted in systemic amphotericin B combined with surgical treatment in only 7 cases. The mortality rate was high (65%) due principally to the delay in diagnosis and absence of surgical treatment.

Isoenzymatic variability of Leishmania infantum in Tunisia concerning 254 human strains.

The different clinical forms of leishmaniasis are the result of both the immunological status of individuals and the species of the parasite causing the infection. In Mediterranean countries, the Leishmania infantum complex groups zymodemes which are responsible for visceral, cutaneous and exceptionally cutaneomucosal or mucosal leishmaniasis. We report in this study a synthesis concerning 254 cases of L. infantum that have been characterized at the "Laboratoire de Parasitologie" of the Rabta Hospital. The strains were isolated from human cases of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) by culture on NNN medium: 156 VL cases and 98 CL cases. The isoenzymatic characterization revealed three zymodemes of L. infantum. * L. infantum MON 1, a common zymodeme of VL,occurred in 154 cases (61%): 147 VL (95%) and 7 CL (5%). All CL cases were from the northern provinces, six of them occurring during an epidemic disease in 2001. * L. infantum MON 24, a common zymodeme of CL in the north, occurred in 98 cases (38.5%): 91 CL (93%) and 7 VL (7%). The seven VL cases were immunocompetent children aged from 8 months to 9 years and native of northern Tunisia. Two of the CL cases were from central regions of the country. This is the first time that cases from these regions are reported. * L. infantum MON 80, an uncommon zymodeme in Tunisia, occurred in two VL cases (0.5%): two children aged 7 and 5. The small number of strains of this zymodeme does not allow understanding of its epidemiological role. The results of this study indicate a low enzymatic variability of L. infantum in the country. However, our study includes only human strains and should be extended to animal ones (dogs, rodents and sand flies). This would lead to a better understanding of the epidemiology of leishmaniasis in Tunisia.

Pediatric visceral leishmaniasis diagnosis in Tunisia: comparative study between optimised PCR assays and parasitological methods.

There has been a steady increase of visceral leishmaniasis during the past 20 years in Tunisia. In this study, we assess the value of two optimised PCR versus those of classical methods for the diagnosis of human visceral leishmaniasis. 106 samples were collected from 53 cases of pediatric visceral leishmaniasis. Peripheral blood and bone marrow samples were analysed both by parasitological methods (direct examination, leukocytoconcentration (LCC) and culture) and by PCR methods with two primer pair (R221/R332 and Lei 70L/Lei 70R). We diagnosed visceral leishmaniasis in all patients: 44 cases were diagnosed by culture (83%), 42 by direct examination of bone marrow (79%), 17 by LCC (32%), and 53 positive cases with both PCR assays (R221/R332 and/or Lei 70L/Lei 70R) (100 %). Regarding each PCR assay, for blood samples, the difference between the sensitivities of PCR Lei 70L/Lei 70R (86,8%) and PCR R221/R332 (17 %) is statistically significant with p-value 0.025. For bone marrow, the sensitivities of the two PCR methods were respectively 96,2% (Lei 70L/Lei 70R) and 75,5% (R221/R332). On the whole, PCR Lei 70L/Lei 70R was more effective than PCR R221/R332 and conventional methods for the two biological samples. Moreover, the requirement of less invasive sample using blood has the advantage of being repeatable for screening and for post therapeutic monitoring.

[Cutaneous leishmaniasis caused by Leishmania infantum MON-24 in Tunisia: extension of the focus to the center of the country]. / La leishmaniose cutanée due a Leishmania infan- tum MON-24 en Tunisie: extension du foyer vers le centre du pays.

Three clinico-epidemiological forms of cutaneous leishmaniasis (CL) were described in Tunisia: the zoonotic CL (ZCL) epidemic which occurred in the centre of the country caused by Leishmania major MON-25, the chronic CL (CCL) In the south-east of the country caused by Leishmania killicki MON-8 and the sporadic CL In the North (SCL) caused by Leishmania infantum MON-24. The latter form, described in 1991, prevails in northern Tunisia with approximately thirty cases per year. Its vector, unknown for a long time could be according to the last publications, Phlebotomus perfiliewi or Phlebotomus langeroni; however, its reservoir remains unknown until now. The systematic isoenzymatic characterization permits to identify a great number of strains improving then knowledge on the eco-epidemiology of the disease. Indeed, changes were noted in the geographical distribution of these clinical forms: extension of the ZCL to the North and South, extension of the CCL to North and the SCL to the centre. We report in this note the first mention of L. infantum MON-24 in the two provinces of the centre of Tunisia: Kairouan and Sidi Bouzid, confirming the extension of the SCL to the Centre.

[Candida dubliniensis: a novel emerging species]. / Candida dubliniensis: une nouvelle espèce émergente.

Over the past decade, the incidence of opportunistic fungal infections continues to increase. Candida albicans remains the most important pathogenic yeast. Since 1995, a novel Candida specie has been identified and named Candida dubliniensis. This specie shares many phenotypic characteristics with Candida albicans, including the ability to produce germ tubes and chlamydospores. These similarities have caused significant problems in the identification of Candida dubliniensis. A large variety of methods have been developed for the discrimination of these two species. They included the sensitivity to different stress (temperature, salinity), growth on chromogenic and specific medium and carbohydrate assimilation. However, these methods can fail to assert the identification of Candida dubliniensis, but molecular typing methods are highly reliable and can confirm the identification. However, they are too complex for routine use. The identification of this specie in routine is essential in order to further understand the epidemiology, to better define the role of Candida dubliniensis as a potential pathogen and its susceptibility to develop a resistance to the antifungal agents. So, new methods have to be developed.

[Onychomycosis in the elderly]. / Onychomycoses chez les sujets âgés.

BACKGROUND: Onychomycosis is more frequent in the elderly. The aim of this Tunisian study was to determine the prevalence and the epidemiological, clinical and mycological characteristics of onychomycosis in elderly subjects. PATIENTS AND METHODS: This was a prospective study performed over a 7-month period (October 2005 - April 2006). The fingernails and toenails of 290 new consecutive patients aged 65 years and over presenting at general practitioners' clinics were examined by certified dermatologists. Clinical signs of onychomycosis were observed in 120 patients. 137 nail samples and 103 skin scrapings were obtained from these subjects. RESULTS: The study population comprised 51 men and 69 women (sex ratio M/F: 0.74) of mean age 72.33 +/- 6.2 years. Fingernails were involved in 28 cases (20.4%), toenails in 109 cases (79.5%), and both fingernails and toenails in 17 cases (12.4%). On average, patients had 2.7 infected fingernails and 3.9 infected toenails. The big toenail and thumbnail were affected most often respectively on the feet and on the hands. Among the clinical presentations seen, distolateral subungual onychomycosis was the most common clinical presentation, on both the feet and the hands. Mycologically confirmed onychomycosis was detected in 110 of 137 nail samples. The rate of positivity was 80.3%. The prevalence of onychomycosis was confirmed in 34.1% of patients examined (99/290). Dermatophytes were the main agents causing onychomycosis in our population (79%). The most frequently detected fungus was Trichophyton rubrum (72.1%). In toenail infections, dermatophytes were most frequently isolated (97%), while in fingernail infections, yeasts were most often isolated (75%), with Candida albicans being dominant (60%). No moulds were isolated in either the hands or the feet. The same fungus was isolated in both skin scrapings and nail samples from 33 patients. Trichophyton rubrum was detected in 28 cases. CONCLUSION: This study confirms a higher prevalence of onychomycosis in the elderly. It shows dermatophytes, in particular Trichophyton rubrum, to be the most frequently isolated causative agents in onychomycosis in the elderly, followed by yeasts, with no moulds being detected.

[Twenty-two cases of neuromeningeal cryptococcosis in Tunisia]. / Vingt-deux cas de cryptococcose neuromeningée en Tunisie.

Neuromeningeal cryptococcosis is a serious infection witch occurs essentially in immunodepressed patients and especially AIDS patients. We report 22 cases of cryptococcosis meningitis confirmed by the parasitology laboratory, in the Tunis Rabta hospital, over a 16-year period. Sixteen patients were HIV infected and six were not HIV infected. The clinical examination documented fever and headache as well as focal neurological signs especially in HIV infected patients. The mycological examination of CSF proved the diagnosis of neuromeningeal cryptococcosis in all cases. The first line treatment was Amphotericin B in 13 cases, Amphotericin B and 5Fluorocytosine in three cases, and fluconazole in six cases. 14 patients died, seven recovered, and one was lost to follow-up.

[Asymptomatic bearing of Leishmania infantum among Tunisian HIV infected patients]. / Portage asymptomatique de Leishmania infantum chez des malades tunisiens infectés par le VIH.

The number of visceral leishmaniasis (VL) cases is in continuous growth in Mediterranean countries. In Tunisia, in addition to the traditional infantile form, more and more cases in immunocompetent or immunocompromised adults have been reported. However, co-infection VL-HIV remains rare in Tunisia and diagnosis of all the cases up till now has been done using traditional techniques (serology, direct examination and culture of bone marrow). However, the last years, several studies proved the greatest sensitivity of PCR in VL diagnosis. We carried out a systematic detection of Leishmania in peripheral blood for 25 HIV infected patients (10 were asymptomatic, 6 presented a fever and/or a paleness and/or an asthenia, and 9 had an opportunist infection other than VL). In all cases, the culture on Novy-Nicolle-McNeal (NNN) medium was negative by the end of the month. Serology carried out for 22 patients was negative in IFI in 17 cases, positive at the 1/20 for four others and positive at the 1/40 for one patient (confirmed by Western Blot technique). A PCR using the primers Lei70L-Lei70R, specific of the gene of Leishmania infantum, allowed the display of the specific band of 345 bp for 17 samples. The higher sensitivity of PCR compared to conventional methods is subject to the difficulty of result interpretation in PCR positive testing among patients not having any other marker of the disease which raises the question of significance for this asymptomatic bearing.

[Which is the role of parasites and yeasts in the genesis of chronic blepharitis?]. / Quel est le rôle des parasites et des levures dans la genèse des blépharites chroniques?

INTRODUCTION: The chronic blepharitis is a current ophthalmic disease posing a therapeutic problem. The etiologic diagnosis is essential for adapting the treatment. PURPOSE: The aim of this study is to describe the role of Demodex sp, Malassezia sp and Candida sp in the pathogenesis of chronic blepharitis. PATIENTS AND METHODS: It's a prospective study realized during a period of 14 months. It interested 69 patients with chronic blepharitis and 96 controls. The prevalence of Demodex sp, Malassezia sp and Candida sp on the eyelash of chronic blepharitis patients and controls was investigated. RESULTS: Demodex folliculorum was found in 58% of patients with chronic blepharitis and in 15,6% of controls. The difference between the two groups was statistically significant (P<0,00001). The sex and the age intervene neither in the frequency, nor in the abundance of D. folliculorum in the two groups. Malassezia sp was identified in 10,4% of the control group and 31,88% of the group with chronic blepharitis. The difference is significant (P<0,00001). There is no correlation between frequency and abundance of Malassezia, sex and age in the two groups. CONCLUSION: In our study, D. folliculorum and Malassezia sp seem to be accused in the genesis of the chronic blepharitis. Their systematic search is necessary in order to adapt the treatment.